J Rubb Res , 6(2), 84-93

Visualisation of the Network Structure in Some

Environmentally Degraded Natural Rubber Gloves
-..* It

M ASRUL #, RT. DAVIES

d-d:

AND A IKRAM

J?

Transmission electron microscopy (TEM) techniques involving swollen rubber samples in a

polymerised matrix were used to characterise the network structure of natural rubber glove pieces,
which had been degraded in soils and in compost TEM analysis of changes in the network
structure of the rubber latex particles was used together with Fourier Transform InfraredAttenuated Total Reflectance (FTIR-ATR) spectroscopy to study various stages of degradation The
extent of degradation was visualised as the breakdown of the rubber latex particles into a loose
network of chain structures, with the accompaniment of considerable reduction in the number of cis
double bonds and the emergence of oxygenated structures These observations however, could
not be related to the actual physical loss in mass of degraded materials, indicating that the
degradation process did not occur uniformly on anv given piece of material left to decompose
Kej words FTIR-ATR, NR, gloves, environment, transmission electron microscopy, network
structure, latex particles, degradation

The natural rubber (NR) glove is one of the

major rubber products derived from a coagulant
dipping process. As a product of a natural
resource, the biodegradabihty of NR gloves
had been described in a number of reports '~6
Adequate work has been done in establishing
the metabolic intermediates of NR biodegradation and it is concluded that the primary
mechanism in NR biodegradation involves
the cleavage of double bonds that affects the
overall structure of a rubber network 14 \
Cook etal8 described a technique invoking
the analysis of stained embedded swollen
samples in a polystyrene matrix using

transmission electron microscopy (TEM).

It was shown that by employing this technique,
the microstructure of the latex film could be
revealed Consequently, the crosslink density
in different types of prevulcamsed latex films
could be compared The extent of degradation
in soil burial tests is usually indicated by
the loss in mass and tensile properties, and
a decline in solvent swelling index. In the
present work, the changes in the microstructure
of gloves degraded in sandy and clayey
soils, and in a composting environment
were observed via network visualisation and
Fourier Transform Infrared-Attenuated Total
Reflectance (FTIR-ATR) spectroscopy

Rubber Research Institute of Malaysia, Malaysian Rubber Board, PO Box 10150, 50908 Kuala Lumpur, Malaysia
* Tun Abdul Razak Research Centre, Brickendonbury, Hertford, SO 13 8NL U K
Corresponding author (e-mail asrul@lgm govmy)

M. Asrul et al: Visualisation of the Network Structure in Some Environmentally Degraded NR Gloves
and left to stand for 2 h to dry after the
extraction. Subsequent drying wras carried out

MATERIAL AND METHODS

Gloves and Treatments

overnight in vacuum at room temperature.

The glove pieces used were from a soil

burial experiment described previously5 and
from a composting experiment.

The extracted glove samples were then

swollen to equilibrium in 0.45 % w/w of
benzoyl peroxide in styrene (99%, Aldrich) and

0.9% di-n-butyl phthalate. Prior to use, the

styrene was washed with sodium hydroxide
(1 M) solution and water to remove the
inhibitor that is present in commercially
available styrene, and then dried over
anhydrous calcium chloride. Diphenylpicryl

In the soil tests, weighed pieces of powdered

examination gloves were buried in unamended
heavy-clay Munchong series (Typic Hapludox)
and a sandy Holyrood series soil (Typic

Quartzipsamment) in large plastic trays and

incubated at 40% field capacity. Gloves
samples retrieved from soils were washed in

hydrazyl (9.1 mg/L styrene) was then added to

running water and dried (60C, 24 h) prior to

provide effective antioxidant protection and to

confer a period of peroxide inhibition prior to

weighing. The pieces retained 64% and 63% of

their initial weights at 12 weeks, respectively,

styrene polymerisation. Portions of the swollen

glove samples in excess styrene were further

for samples from the clayey and sandy soils,

heated in a gelatine capsule at 68C for 48 h or

until the styrene had fully polymerised.

and 21% and 57% at 24 weeks.

In the composting experiment, the NR glove
pieces were placed in nylon net (38 um pore
size) mesh bags within a compost mix

Transmission Electron Microscopy

developed from dried grass clippings, cattle

manure and soil (6:3:1, v/v). The compost

Ultra-thin sections (= 100-150 nm) were cut

from the swollen embedded samples at -110C

with an RMC MT-7000 ultramicrotome using
45 freshly cleaved glass knives set at a shallow-

pile was maintained at 40% moisture within

43 X 51 (D X H) cm3 PVC compost bins, and
the pile turned over manually at seven-day

clearance angle. The sections obtained were

intervals. Pile temperatures were monitored

daily, and the moisture adjusted weekly to
prevent inhibition of microbial activity. The
composting process was operated for 24 weeks.
The pieces from the composting pile had 55%

placed on grids that had previously been dipped

in ethanol in order to aid sample collection.
The sections were stained with osmium
tetroxide vapour for one hour. The stained
sections were then examined using a Philips

and 27% of their initial weights remaining at

EM 300 Transmission Electron Microscope

12 weeks and 24 weeks.

operating at 100 kV with a magnification of

36 000 times.

Preparation of
Visualisation

Samples for Network

FT-IR Spectroscopy

The glove samples were continuously

extracted with cold acetone (Analar) in a
Soxhlet apparatus for 24 h at room temperature

The spectra of the glove surfaces were

obtained using a Perkin-Elmer Spectrum 2000
FTIR spectrophotometer with ATR accessory

COPYRIGHT MALAYSIAN RUBBER BOARD

85

Journal of Rubber Research, Volume 6(2), 2003

(Specac MK II Golden Gate Ge-ATR). A total

the gloves, which had been degraded in soil,

of 50 scans were accumulated with a resolution

of 4 cm '.

nearly all of this latex particle structure was

destroyed (Figure 2). Also, in the undegraded

glove the particles were linked fairly closely

together by a loose network of rubber threads,

but in the degraded gloves large areas of

RESULTS

polystyrene infiltrated the weaknesses in the

overall structure of the rubber network.

Network-visualisation

Osmium tetroxide preferentially stains

In the case of the gloves, which had undergone composting, more of the latex particle

carbon-carbon double bonds; the dark phase in

the micrographs is rubber and the light phase is
polystyrene. In an undcgraded glove, swollen
individual rubber latex particles appeared as
dark areas of threadlike mesh with distinct

structures were retained although several

regions exhibited a low network density where

large amounts of polystyrene were visible

(Figure 3).

boundaries surrounded by white areas of

The 24-week-old glove sample

displayed more areas of a low network density

than the 12-week-old sample.

polystyrene matrix (Figure 1). By contrast, in

Figure I . TEM micrograph of undegraded NR gloves (Bars = 0.5 fan).

86

Figure 2 TE\lmicrogfaph of \Rglo\ts in sand\ and c/aui

4 and B 12 and 24 week-old sample?, in clayey soil
C and D 12 and 24 \\eek old samples in sandy soils (Bar\ =05 fim)

figure ? TEMmicrograph of \R g!o\es in compost

E and F 12 and 24 \\eek-old sampler fBan = 0

M Asrul et al Visualisation of the Network Structure in Some Environmentally Degraded NR Gloves

Analysis b\ FTIR-ATR

ments could be earned out on severely degraded

Samples after degradation showed the

appearance in oxygenated structures particularly
in the region of 1780 cm ] to 1630 cm ' that
could be attributed to carbonyl groups and at

NR specimens Previous work has shown that

the mesh sizes of different types of styreneswollen dry rubbers could be corielated to the
molecular weight between crosslinks as
determined by stress-strain measurements12,

1070 cm ' to 1030 cm ' that corresponded to

the formation of primary alcohols (Figure 4}

and that vulcamsates with a high crosslink

density produce finer and smaller mesh sizes

Disappearance of the =C-H out of plane band

at 835 cm ' were also observed for all the
biodegraded NR gloves compared to that of the
controls These observations corresponded to

than vulcamsates with a lower crosslink

density Amir-Hashim et al R found the same
effect when using a similar technique to
investigate the effect of leaching m NR latex

oxidative chain scissions, as the formation of

films

oxygenated structures such as ketones and

aldehydes is common for oxidative chain
scission m NRy, or could also be interpreted
as oxidative reduction arising from NR
biodegradation"

The results for the glove samples buried in

sandv and clayey soils were similar in the
trends observed. / e the relative disappearance
of the us double bond and the occurrence of
oxygenated structures in the 24-week-old
samples were appreciably higher than the
12-week-old samples
A difference was
observed with the 24-week-old samples of
gloves in compost, where the primary alcohols
and carbonyl group bands were comparative!}
lower than the 12-vveek-old sample (Figure 5}

Both degraded samples m compost also

appeared to retain the band attributed to the

=C-H out of plane band at 835 cm ' and

another major band of MR at 1450 cm '
corresponding to the CH2 deformation"

In the present work it was found that after

degradation in soil the integrity of the network
structure of the glove samples was destroyed
and therefore the polystyrene was able to

infiltrate the latex particle micro structure A

high degree of this phase separation between
the rubber and the polystyrene indicated that a
high degree of degradation had taken place and
that the strength of the forces maintaining
the integrity of latex film had diminished
However, some areas of high density rubber

network could still be observed in the samples

and these presumably represented regions of
highly crosslmked rubber

The degree of phase separation between the

rubber and the polystyrene that occurred was
similar for the gloves degraded in the clayey
and sand} soils
However, in NR gloves
degraded in compost the micro structures of the

latex particles were still present and visible

after 12 and 24 weeks In addition, the FTIR-

ATR spectra of the 12 and 24-week-old glove

DISCUSSION

Netw ork-vi suahsation

of

styrenc-swollen

samples in compost appeared to retain bands

attributed to NR, namely the t n double bond
and CH2 deformation

samples of degraded gloves may provide a way

to studv. the various stages of the biodegradation process since alternative physical testing
methods such as tensile or swelling measure-

Although such results mav indicate that

degradation in compost was less pronounced

compared to degradation in soils, the situation

(a) Sandy soil

835.963

2000

1800

1600

1400

1200

1000

800

700

Wave-number (cm~l)

(b) Sandy soil

835.963

2000

1800

1600

1400

1200

1000

Wave-number (cm-')

Figure 4. FTIR-ATR analysis of degraded NR gloves.

800

700

M. Asrul et al: Visualisation of the Network Structure in Some Environmentally Degraded NR Gloves

835.963

-eo

2000

1800 1600 1400 1200 1000

Wave-number (cm-1)

800

700

Figure 5. FTIR-ATR analysis of degraded NR gloves in compost.

was unlikely since the composting process

provides favourable environmental conditions
(increased microbial activity, nutrient availability, adequate oxygen, optimal moisture
retention for microbial activity, early
thermophilic stage) that enhance the rate of
natural decomposition. Furthermore, the
severity of the breakdown observed did not
reflect the actual physical loss in mass of the
degraded materials. For example, the sandy
soil glove samples retained 21% of their initial
weights (79% weight loss) at 24 weeks

compared to 57% (43% weight loss) for the

clayey soil, despite their similar micrographs.
Similarly, glove pieces from compost had 55%
and 27% of their initial weights remaining at
12 and 24 weeks, but the state of their network
mesh structure suggested an early stage of
degradation.
These findings indicate that the biodegradation process was not proceeding uniformly
throughout any given piece of material left to
decompose. It is likely that biodegraded
91

Journal of Rubber Research, Volume 6(2), 2003

shorter fragment of rubber chains were assimilated into surrounding soils, accounting for the
mass losses, and leaving behind intact and highly
crosshnked materials However, the presence of
microstructures of the latex particles that
could still be observed m the gloves samples

3 LINOS, A BEREKAA, MM REICHEET,

R , KEEEER, U, SCHMITT, J, FEEMMING, H , KROPPENSTEDT, RM AND
STEINBUCHEE, A (2000) Biodegradation
of cw-pol>isoprene Rubbers by Distinct

Actmomycetes Microbial Strategies and

Detailed Surface Analysis Applied and
Environmental Microbiology, 66, 1639-1645

degraded in compost remained unclear

4 BODE, H B , ZEECK, A , PLUCKHAHN,

K AND JENDROSSEK, D (2000)

CONCLUSION

Physiological and Chemical Investigations

into Microbial Degradation of Synthetic

The present investigation demonstrates the

disruption of the visualised rubber network
structures and the loss of NR as double bonds
following environmental degradation It
could be deduced that degradation was not

Poly(czs-l 4-isoprene) Applied and Environmental Microbiology, 66, 36803685

5 IKRAM, A , AEIAS O AND NAP1, D (2000)
Biodegradabihty of NR Gloves in Soils J
Rubb Res, 3(2), 104-114

progressing uniformly on gloves samples left to

degrade as the -visualisation of the network

structures could not be correlated to the sample
weight loss

6 GALEERT, C (2000) Degradation of Latex

and of Natural Rubber by Streptomvces
Strain La 7 Systemic and Applied
Microbiology, 23, 433-441

ACKNO\V LEDGEMENTS
7 TSUCHII A AND TAKEDA, K

(1990)

Rubber-degrading Enzyme from a Bacterial

The authors would like to thank Kathenne

Lawrence of the Material Characterization

Culture
Applied and Environmental
Microbiolog\, 56, 269-274

Section of TARRC for preparing samples for

TEM

8 COOK, S , CUDBY P E E DAVIES, RT

AND MORRIS, MD
(1997) The
Microstructure of Natural Rubber Films
Rubb Chem Technol, 70, 549-559

Date of receipt \ovember 2002

Date of acceptance Vfay 2003

9 BARNARD, D AND LEWIS PM (1988)

Oxidative Ageing, Natural Rubber Science
and Technology (Roberts AD ed), p
621-678 Oxford Oxford University Press

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2 HEISEY, RM AND PAPADATOS S (1995)

Isolation of Microorganisms Able to
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KAWASAKI H (1998) Reaction of Moist

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M. Asrul et al: Visualisation of the Network Structure in Some Environmentally Degraded NR Gloves
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